Francisella tularensis is a zoonotic bacterial pathogen that causes fatal infections in hundreds of different animal species. Human infections result from transmission by insect vectors, direct physical contact with infected animals, ingestion of contaminated food or water, and inhalation of aerosolized organisms. All contact routes can result in disseminated infections, but the highest morbidity and mortality is associated with inhalation [unreadable] initiated infection. Our goal is to understand the host pathogen interactions that define pulmonary tularemia. Towards that end we have developed in vivo and in vitro models to study F. tularensis interactions with different lung cell types. With these models we have found that F. tularensis invades and replicates within tissue and alveolar macrophages, dendritic cells, neutrophils and alveolar type II (AT-II) epithelial cells following inhalation. Invasion and replication within AT-II cells represents a novel environmental niche for a bacterial pathogen. In Aim 1 we will characterize F. tularensis interactions with AT-II cells, focusing on bacterial trafficking and its impact on AT-II cell physiology. A transposon insertion mutant library was screened to identify strains that were deficient for replication within AT-II cells. One such mutant had a transposon insertion in a gene we termed regA. The regA gene sequence was unique to Francisella species. RegA expression was induced 180-fold upon entry into AT-II cells, and 6-fold upon entry into J774 cells. The transposon mutant invaded but failed to replicate in AT-II cells, however it retained the ability to replicate within macrophages, whereas regA deletion mutants did not replicate in either AT-II cells or macrophages. The expression of a number of genes was altered in both deletion and insertion regA mutant strains as determined by microarry and 2D-gel analysis. . Five of the genes affected by RegA were induced in wild type bacteria grown in TC-1 cells, and were also identified in other genetic screens for virulence-associated genes. Based on these and other data we propose that RegA is involved in the sensing and adaptation response to the intracellular environment. In Aim 2 we will examine the properties and function of, RegA, and determine the mechanism by which its expression is regulated. In Aim 3, 5 loci encoding proteins with unknown function and whose expression is affected by RegA will be subjected to genetic and biochemical analysis to determine their roles in Francisella intracellular growth.